This proposal is part of an effort to understand how cells localize molecules and organelles to specific regions of the cytoplasm, which is referreed to as polarization. The observation that gradients of intracellular calcium ions ((Ca2+)i) can form in cells represents both an example, and perhaps, a clue to the solution of the problem. The cytotoxic T lymphocyte (CTL), is presently being used as a model system for thses studies. The CTL is important in itself as an important arm of the immune system, and an excellent system for the study of cell polarization. The CTL exhibits polarization of organelles in response to binding of appropriate target cells. At the time these movements are taking place, a calcium gradient also forms. Since calcium is an important regulatory element and is known to affect molecules invovled in cell motility, it is likley that the two are related. The aim of this project is to understand the mechanism of (Ca2+)i gradient formation and how Ca2+ might act locally to polarize the cell. A model has been formulated to explain the formation of a (Ca2+)i gradient which involves the participation of another important regulatory molecule, protein kinase C. It appears that protein kinase C is also locally activated in CTLs with a polarity opposite that of the calcium gradient. Since protein kinase C can in some cases act to lower (Ca2+)i it may be part of the mechanism for forming the (Ca2+)i gradient. This study will test the hypothesis that local activation of kinase C can activate calcium pumping systems and locally lower (Ca2+)i resulting in a (Ca2+)i gradient. Finally, since (Ca2+)i and protein kinase C can have reciprocal effects on the activity of some proteins, a Foundation is laid for polarity. This study will attempt to detect localized differences in the phosphorylation state of enzymes that are modulated reciprocally by (Ca2+)i and protein kinase C.